Journal: Cell Death & Disease

Article Title: Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways

doi: 10.1038/cddis.2014.247

Figure Lengend Snippet: G93A-SOD1 toxicity is not mediated by p53 acetylation state or by IRS-2/Ras/ERK1/2 pathway in SH-SY5Y cells. ( a ) Western blot analysis of 20 μ M of total protein extract from cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) and treated with 3 μ M Ex527 or DMSO. Antibodies against SIRT1, Erk1/2, pErk1/2, p53, p53-Ac, tubulin and Ac-tubulin were used. β -Actin was used as loading control. One representative blot is shown from three independent experiments giving comparable results. ( b ) Densitometric analysis of results as in ( a ); data are expressed as acetylation ratio of p53 and tubulin. ( c ) Cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) were treated with 3 μ M Ex527 or with 3 μ M SL327 or both. Cell viability was assessed and reported for . Values significantly different from relative controls are indicated with * P <0.05 with respect to Ctrl and # P <0.05 with respect to DMSO. ( d ) Western blot analysis of 20 μ M of total protein extract from C2C12 cells differentiated for 5 days and expressing G93A-SOD1 (G93A) or untransfected (Ctrl). Antibodies against SIRT1, p53 and p53-Ac were used. β -Actin was used as loading control. One representative blot is shown from three independent experiments giving comparable results. ( e ) Densitometric analysis of data as in ( d ); data are expressed as acetylation ratio of p53

Article Snippet: AGK2, Ex527, Sirtinol, SRT1720 and SL327 were from Tocris Bioscience (Bristol, UK); all of them were resuspended in an appropriate volume of DMSO and kept stored at −80°C before use.

Techniques: Western Blot, Infection, Control, Expressing

Journal: Molecular cell

Article Title: An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells

doi: 10.1016/j.molcel.2019.05.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Biotinylated lectins (Vector Laboratories, Burlingame, CA) were incubated at different concentrations for 1 h, followed by washing and incubation with Streptavidin-Alexa Fluor488 (Invitrogen) for 1 h. Recombinant human Siglec Fc-chimera (R&D systems, Minneapolis, MN) were precomplexed with Alexa Fluor647 goat anti-human IgG antibody (Life Technologies) for 1 h followed by incubation with cells for 1 hr.

Techniques: Recombinant, Plasmid Preparation, Construct, Software

Journal: Cancer research

Article Title: Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression

doi: 10.1158/0008-5472.CAN-18-2931

Figure Lengend Snippet: ASPN binds to BMP-4 and restricts BMP-4-induced signaling. A, GSEA of TGF-β family pathway genes in Aspn+/+ and Aspn−/− fetal MSCs as determined from microarray (n=3). B, C, BMP-4-induced signaling in Aspn+/+ and Aspn−/− fetal MSCs (A and B represent independently derived MSCs) as measured by immunoblotting and quantified using ImageJ. Aspn+/+ and Aspn−/− fetal MSCs were serum-depleted and then incubated with recombinant mouse BMP-4 (5ng/mL) for 30 minutes. Statistical analyses performed using Student’s t-test (n≥3). D, E, Recombinant mouse ASPN restricts BMP-4-induced signaling in Aspn−/− fetal MSCs as measured by immunoblotting and quantified using ImageJ. Aspn+/+ and Aspn−/− fetal MSCs were serum-depleted and then incubated with recombinant mouse BMP-4 (5ng/mL) with vehicle or recombinant mouse ASPN (100ng/mL) for 30 minutes. Statistical analyses performed using Student’s t-test (n≥2). F, G, BMP-4-induced signaling in WPMY-1-Neo, WPMY-1-ASPN D13, and WPMY-1ASPN D14 (A and B represent independently derived clones) as measured by immunoblotting and quantified using ImageJ. WPMY-1-Neo, WPMY-1-ASPN D13, and WPMY-1ASPN D14 were serum-depleted and then incubated with recombinant human BMP-4 (5ng/mL) for 30 minutes. Statistical analyses performed using one-way ANOVA with Tukey multiple comparison (n≥3). H, Co-immunoprecipitation of ASPN and BMP-4. FLAG tagged mouse ASPN was transfected in HEK293T cells and then incubated with recombinant mouse BMP-4. ASPN was immunoprecipitated with anti-FLAG beads and then examined by immunoblotting for ASPN and BMP-4 (n=2). I, Aspn+/+ and Aspn−/− fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250nM LDN-193189, or recombinant mouse BMP4 (5ng/mL) and then stained for Alizarin Red. J, Aspn+/+ and Aspn−/− fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250nM LDN-193189, or recombinant mouse BMP4 (5ng/mL) and then examined for osteogenic-induced genes by qRT-PCR. Statistical analyses performed using one-way ANOVA with Tukey multiple comparison (n≥3). Graphs shown as mean±SEM, *P≤0.05, **P≤0.01, ***P≤0.001, and black bars=100μM.

Article Snippet: Co-immunoprecipitation assays HEK293T cells were transfected with a murine ASPN-Myc-FLAG tagged expression vector (Origene) or vehicle control using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Microarray, Derivative Assay, Western Blot, Incubation, Recombinant, Clone Assay, Immunoprecipitation, Transfection, Cell Culture, Staining, Quantitative RT-PCR

Journal: Cancer research

Article Title: Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression

doi: 10.1158/0008-5472.CAN-18-2931

Figure Lengend Snippet: Key Resources

Article Snippet: Co-immunoprecipitation assays HEK293T cells were transfected with a murine ASPN-Myc-FLAG tagged expression vector (Origene) or vehicle control using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Magnetic Beads, Recombinant, Transfection, Staining, Generated, Blocking Assay, Bicinchoninic Acid Protein Assay, Cell Migration Assay, Mutagenesis, Random Hexamer Labeling, Expressing, Software

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: NETRIN-1 expression was significantly reduced, whereas SLIT3 expression was markedly elevated in the corneal epithelium of Aqp5 −/− mice compared to Aqp5 +/+ mice. ( A ) Heatmap illustrates differentially expressed neurotrophic and axon guidance factors in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( B ) Volcano plot analysis of differentially expressed neurotrophic and axon guidance factors between the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice, with upregulated genes highlighted in red and downregulated genes in blue . ( C ) Quantitative RT-PCR analysis of Ntn1 and Slit3 expression levels in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice ( n = 3). ( D ) Representative immunofluorescence images of corneas stained with NETRIN-1 and SLIT3 ( green ) and DAPI ( blue ). Scale bars : 20 µm. ( E ) Regional quantification of positive immunofluorescence staining from images in ( D ) ( n = 3). ( F ) Western blot analysis of NETRIN-1, SLIT3, and ROBO4 protein expression in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( G ) Statistical evaluation of Western blot data ( n = 6). ( H ) Immunofluorescence staining of primary corneal epithelial cells isolated from Aqp5 +/+ and Aqp5 −/− mice, labeled with DAPI ( blue ), NETRIN-1, and SLIT3 ( green ). Scale bars : 25 µm. ( I ) Regional quantification of positive immunofluorescence staining from images in ( H ) ( n = 6). ( J ) The protein concentration of NETRIN-1 and SLIT3 in primary corneal epithelial cell supernatant was detected through ELISA ( n = 3). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant NETRIN-1 (4 μg/mL; #1109-N1; R&D Systems, Minneapolis, MN, USA), SLIT3 (4 μg/mL; #9296-SL; R&D Systems), corneal epithelial extracts, or neutralizing antibodies against NETRIN-1 (4 μg/mL; #AF1109; R&D Systems) and SLIT3 (4 μg/mL; #AF3629; R&D Systems) were added for 48 hours to assess their effects on axon growth.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot, Isolation, Labeling, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: NETRIN-1 and SLIT3 affected neuronal axon growth. ( A ) Axonal growth status of Aqp5 +/+ and Aqp5 −/− TG neurons following treatment with recombinant mouse NETRIN-1 (rmNETRIN-1) and recombinant mouse SLIT3 (rmSLIT3). Scale bars : 50 µm. ( B ) Statistical analysis of the maximum branch lengths of TG neurons ( n = 6). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant NETRIN-1 (4 μg/mL; #1109-N1; R&D Systems, Minneapolis, MN, USA), SLIT3 (4 μg/mL; #9296-SL; R&D Systems), corneal epithelial extracts, or neutralizing antibodies against NETRIN-1 (4 μg/mL; #AF1109; R&D Systems) and SLIT3 (4 μg/mL; #AF3629; R&D Systems) were added for 48 hours to assess their effects on axon growth.

Techniques: Recombinant

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: Effects of corneal epithelial TEs and NAbs on axon growth in TG neurons. ( A ) Left : Axon growth was assessed following the addition of Aqp5 −/− and Aqp5 +/+ corneal epithelial TEs to Aqp5 +/+ TG neurons. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons shown in ( A , i ) ( n = 6). ( B ) Left : Axon growth was assessed following the addition of Aqp5 −/− and Aqp5 +/+ corneal epithelial TEs to Aqp5 −/− TG neurons. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons shown in ( B , i ) ( n = 6). ( C ) Left : Axon growth following the simultaneous addition of Aqp5 −/− TEs or SLIT3 NAbs. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons presented in ( C , i ) ( n = 6). ( D ) Left : Axon growth following the simultaneous addition of Aqp5 +/+ TEs or NETRIN-1 NAbs. Scale bars : 50 µm. Right : Statistical analysis of the maximum branch length of axons shown in ( D , i ) ( n = 6). Results of statistical analyses are expressed as mean ± SD. *** P < 0.001.

Article Snippet: Recombinant NETRIN-1 (4 μg/mL; #1109-N1; R&D Systems, Minneapolis, MN, USA), SLIT3 (4 μg/mL; #9296-SL; R&D Systems), corneal epithelial extracts, or neutralizing antibodies against NETRIN-1 (4 μg/mL; #AF1109; R&D Systems) and SLIT3 (4 μg/mL; #AF3629; R&D Systems) were added for 48 hours to assess their effects on axon growth.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: AQP5 Regulates Corneal Nerve Distribution Through NETRIN-1 and SLIT3

doi: 10.1167/iovs.66.14.6

Figure Lengend Snippet: The transcription factor JUN-D bound to and regulated the expression of NETRIN-1 and SLIT3 in corneal epithelial cells. ( A ) Heatmap analysis of Ntn1 and Slit3 cotranscription factor expression levels in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( B ) Western blot analysis of JUN-D protein expression levels in the corneal epithelium of Aqp5 +/+ and Aqp5 −/− mice. ( C ) Quantitative statistical analysis of Western blot data ( n = 3). ( D ) ChIP-PCR analysis revealed significant enrichment of JUN-D in the promoter regions of Ntn1 and Slit3 . ( E ) Quantitative RT-PCR analysis was conducted to evaluate the expression levels of Ntn1 and Slit3 in corneal epithelial cells isolated from Aqp5 +/+ mice following 24-hour treatment with SR11302 ( n = 3). ( F ) Western blot analysis of NETRIN-1 and SLIT3 protein expression in the corneal epithelial cells of Aqp5 +/+ mice treated with SR11302. ( G ) Quantitative statistical evaluation of Western blot data ( n = 6). Statistical results are presented as mean ± standard deviation (SD). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant NETRIN-1 (4 μg/mL; #1109-N1; R&D Systems, Minneapolis, MN, USA), SLIT3 (4 μg/mL; #9296-SL; R&D Systems), corneal epithelial extracts, or neutralizing antibodies against NETRIN-1 (4 μg/mL; #AF1109; R&D Systems) and SLIT3 (4 μg/mL; #AF3629; R&D Systems) were added for 48 hours to assess their effects on axon growth.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Isolation, Standard Deviation